Comprehensive Analysis of the Secreted Proteins of the Parasite Haemonchus contortus Reveals Extensive Sequence Variation and Differential Immune Recognition
2003; Elsevier BV; Volume: 278; Issue: 19 Linguagem: Inglês
10.1074/jbc.m212453200
ISSN1083-351X
AutoresAna Patrícia Yatsuda, Jeroen Krijgsveld, Albert W.C.A. Cornelissen, Albert J. R. Heck, Erik de Vries,
Tópico(s)Parasites and Host Interactions
ResumoHaemonchus contortus is a nematode that infects small ruminants. It releases a variety of molecules, designated excretory/secretory products (ESP), into the host. Although the composition of ESP is largely unknown, it is a source of potential vaccine components because ESP are able to induce up to 907 protection in sheep. We used proteomic tools to analyze ESP proteins and determined the recognition of these individual proteins by hyperimmune sera. Following two-dimensional electrophoresis of ESP, matrix-assisted laser desorption ionization time-of-flight and liquid chromatography-tandem mass spectrometry were used for protein identification. Few sequences of H. contortus have been determined. Therefore, the data base of expressed sequence tags (dbEST) and a data base consisting of contigs fromHaemonchus ESTs were also consulted for identification. Approximately 200 individual spots were observed in the two-dimensional gel. Comprehensive proteomics analysis, combined with bioinformatic search tools, identified 107 proteins in 102 spots. The data include known as well as novel proteins such as serine, metallo- and aspartyl proteases, in addition to H. contortus ESP components like Hc24, Hc40, Hc15, and apical gut GA1 proteins. Novel proteins were identified from matches with H. contortus ESTs displaying high similarity with proteins like cyclophilins, nucleoside diphosphate kinase, OV39 antigen, and undescribed homologues ofCaenorhabditis elegans. Of special note is the finding of microsomal peptidase H11, a vaccine candidate previously regarded as a "hidden antigen" because it was not found in ESP. Extensive sequence variation is present in the abundant Hc15 proteins. The Hc15 isoforms are differentially recognized by hyperimmune sera, pointing to a possible specific role of Hc15 in the infectious process and/or in immune evasion. This concept and the identification of multiple novel immune-recognized components in ESP should assist future vaccine development strategies. Haemonchus contortus is a nematode that infects small ruminants. It releases a variety of molecules, designated excretory/secretory products (ESP), into the host. Although the composition of ESP is largely unknown, it is a source of potential vaccine components because ESP are able to induce up to 907 protection in sheep. We used proteomic tools to analyze ESP proteins and determined the recognition of these individual proteins by hyperimmune sera. Following two-dimensional electrophoresis of ESP, matrix-assisted laser desorption ionization time-of-flight and liquid chromatography-tandem mass spectrometry were used for protein identification. Few sequences of H. contortus have been determined. Therefore, the data base of expressed sequence tags (dbEST) and a data base consisting of contigs fromHaemonchus ESTs were also consulted for identification. Approximately 200 individual spots were observed in the two-dimensional gel. Comprehensive proteomics analysis, combined with bioinformatic search tools, identified 107 proteins in 102 spots. The data include known as well as novel proteins such as serine, metallo- and aspartyl proteases, in addition to H. contortus ESP components like Hc24, Hc40, Hc15, and apical gut GA1 proteins. Novel proteins were identified from matches with H. contortus ESTs displaying high similarity with proteins like cyclophilins, nucleoside diphosphate kinase, OV39 antigen, and undescribed homologues ofCaenorhabditis elegans. Of special note is the finding of microsomal peptidase H11, a vaccine candidate previously regarded as a "hidden antigen" because it was not found in ESP. Extensive sequence variation is present in the abundant Hc15 proteins. The Hc15 isoforms are differentially recognized by hyperimmune sera, pointing to a possible specific role of Hc15 in the infectious process and/or in immune evasion. This concept and the identification of multiple novel immune-recognized components in ESP should assist future vaccine development strategies. gastrointestinal molecular weight isoelectric point excretory/secretory ES products phosphate-buffered saline 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid cyclophilins expressed sequenced tag data base of EST mass spectrometry matrix-assisted laser desorption ionization time-of-flight liquid chromatography-tandem mass spectrometry Gastrointestinal (GI)1nematodes are currently controlled by the use of chemicals (anti-helminthics), but there is great interest in development of vaccines, mainly because of the emergence of drug-resistant parasites. Among the GI nematodes, Haemonchus contortus is economically important because of its blood feeding characteristics in the abomasum; it is able to cause severe losses in production of small ruminant herds. The parasitic stages of the developmental cycle occur in a single host, and during each phase the nematode releases a variety of molecules into the host or in vitro culture environment. These are usually referred to as excretory/secretory products (ESP). ESP are of practical value as a source of potential vaccine components. Obtained after in vitro cultivation of adult worms in serum-free medium, ESP or its partially purified fractions are able to induce 65 to 907 protection against H. contortus in sheep (1Schallig H.D. van Leeuwen M.A. Cornelissen A.W. Parasite Immunol. 1997; 19: 447-453Crossref PubMed Scopus (84) Google Scholar, 2Vervelde L. Van Leeuwen M.A. Kruidenier M. Kooyman F.N. Huntley J.F. Van Die I. Cornelissen A.W. Parasite Immunol. 2002; 24: 189-201Crossref PubMed Scopus (34) Google Scholar), but protective properties have not conclusively been attributed to individual proteins. Regardless of its practical use in vaccination studies, there is hardly any conclusive evidence on the biological function of ESP. For some proteins, functional roles have been proposed on the basis of similarity to other proteins in sequence data bases (3Loukas A. Doedens A. Hintz M. Maizels R.M. Parasitology. 2000; 121: 545-554Crossref PubMed Scopus (63) Google Scholar, 4Gems D. Ferguson C.J. Robertson B.D. Nieves R. Page A.P. Blaxter M.L. Maizels R.M. J. Biol. Chem. 1995; 270: 18517-18522Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), or roles in immune evasion or in molting have been implied (5Loukas A. Mullin N.P. Tetteh K.K. Moens L. Maizels R.M. Curr. Biol. 1999; 9: 825-828Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 6Hong X. Bouvier J. Wong M.M. Yamagata G.Y.L. McKerrow J.H. Exp. Parasitol. 1993; 76: 127-133Crossref PubMed Scopus (59) Google Scholar). Furthermore, the surface of the cuticle may be constituted of proteins derived from the secretory system (7Bird A.F. Bird J. The Structure of Nematodes. 2nd Ed. Academic Press Ltd., London1991: 167-229Crossref Google Scholar). The secretion of some proteins has been linked in time to the transition of free-living stages to parasitism (8Hawdon J.M. Jones B.F. Hoffman D.R. Hotez P.J. J. Biol. Chem. 1996; 271: 6672-6678Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). The identification of the proteins present in ESP of H. contortus using a proteomics approach will provide a basis for studies on the following matters. (i) Identification of ES proteins as the products of specific genes, enabling bioinformatic analyses and much more specific functional studies. (ii) The complexity of ES, which has hardly been addressed by the almost exclusive use of one-dimensional gel electrophoresis. (iii) The variability between batches of ESP, a topic highly relevant to vaccination studies. (iv) The recognition of specific spots by immune sera, especially in cases where multiple spots are derived from the expression of multigene families or from variations in post-translational modifications. (v) The cellular origin of ESP. Among others, ESP can contain proteins secreted by the pharyngeal glands, the excretory system, epithelial cells of the intestine (e.g. digestive enzymes), or rectal and vaginal cells (7Bird A.F. Bird J. The Structure of Nematodes. 2nd Ed. Academic Press Ltd., London1991: 167-229Crossref Google Scholar). Moreover, cytosolic components of decaying cells (either by apoptosis or other causes of damage) may be present in addition to epithelial membrane proteins that are cleaved off. Here we have described 107 identifications in the ESP of adults ofH. contortus from gene sequence information deposited in GenBankTM nr as well as from H. contortus dbEST tag sequences. A number of the identified proteins have previously been associated with a protective immune response, although in several cases their presence in ESP has not been reported. For many proteins we have now shown that they appear in truncated forms or have extensive sequence modifications, which may complicate the design of vaccination strategies. Furthermore, many proteins have been detected in ESP for the first time and will be discussed with regard to their potential function and relation to an immune response. Standard procedures for harvesting ESP have been used as described for H. contortus and other nematodes (3Loukas A. Doedens A. Hintz M. Maizels R.M. Parasitology. 2000; 121: 545-554Crossref PubMed Scopus (63) Google Scholar, 9Schallig H.D.F.H. van Leeuwen M.A.W. Hendrikx W.M.L. Parasitolology. 1994; 108: 351-357Crossref PubMed Scopus (85) Google Scholar, 10Holland M.J. Harcus Y.M. Riches P.L. Maizels R.M. Eur. J. Immunol. 2000; 30: 1977-1987Crossref PubMed Scopus (118) Google Scholar, 11Healer J. Ashall F. Maizels R.M. Parasitolology. 1991; 103: 305-314Crossref PubMed Scopus (34) Google Scholar, 12Chandarshekar R. Curtis K.C. Li B.W. Weil G.J. Parasitology. 1995; 73: 231-239Google Scholar, 13Todorova V.K. Knox D.P. Kennedy M.W. Parasitology. 1995; 111: 201-208Crossref PubMed Scopus (49) Google Scholar). All batches (A to D) were derived from experimental infections initiated from different larval stocks established over a period of 2 years from new generations of the same isolate. H. contortus adult worms were harvested from the abomasum of infected donor lambs, washed several times in PBS, and kept in RPMI 1640 medium containing antibiotics (100 IU of penicillin, 0.1 mg/ml streptomycin, and 5 ॖg/ml gentamicin) at 37 °C under 57 CO2. The parasites were first incubated for 4 h, after which the medium was harvested and new medium containing 27 glucose was added for overnight incubation. The supernatant was collected, centrifuged, filter-sterilized (0.2 ॖm), concentrated, and desalted (10 mm Tris, NaCl pH7.4) in 3-kDa filters (Centripep YM-3, Millipore). Prior to isoelectric focusing, the ESP were precipitated in a final concentration of 107 trichloroacetic acid (dissolved in acetone) containing 10 mm dithiothreitol. The pellet was washed one time in acetone with 10 mm dithiothreitol and resuspended in rehydration solution (7 m urea, 2m thiourea, 47 CHAPS, 27 carrier ampholyte mixture, pH 3–10NL (IPG buffer) and 20 mm dithiothreitol). Isoelectric focusing instrumentation, IPG gels, and related reagents were fromAmersham Biosciences unless otherwise indicated. Either 70 or 140 ॖg of protein was loaded onto 13-cm IPG strips (pH 3–10NL) and supplemented with protease inhibitors (Complete Protease Inhibitor mixture, Roche Molecular Biochemicals). The sample was rehydrated and focused in an automated overnight run (IPGPhorTM) using 10–14 h of rehydration (30 V), followed by a step voltage focusing procedure (1 h 500 V, 1 h 1000 V followed by 8000 V until a total of 35–40 Kvh was reached). The strips were incubated in 10 ml of equilibration buffer (50 mm Tris, 6m urea, 27 SDS, 307 glycerol, pH 8.8) containing 30 mm dithiothreitol for the first 15 min and replaced by equilibration buffer with 135 mm iodoacetamide for another 15 min. Electrophoresis in second dimension gel SDS-gel was carried out in a Hoefer SE600 system. Silver staining or Coomassie Brilliant Blue R-250 was used to visualize proteins after second dimensional electrophoresis. The images of the gels were acquired using LabScan v3.0 software on an ImageScanner (Amersham Biosciences). ESP of H. contortus were separated in 12.57 SDS-PAGE gels and transferred to polyvinylidene difluoride membranes (ImmobilonP, Millipore) using a semi-dry system (Novablot, Hoefer) in transfer buffer (39 mm glycine, 48 mm Tris, 0.03757 SDS, 207 methanol) at 1.1 mA/cm2 for 1 h. The transfer efficiency was checked by staining of the membranes with DB71 (14Hong H.Y. Yoo G.S. Choi J.K. Electrophoresis. 2000; 21: 841-845Crossref PubMed Scopus (42) Google Scholar). The membranes were blocked with 57 skimmed milk in PBS/0.057 Tween 20 (PBS-T, 1h, 37 °C) and all the washing steps were done with PBS-T (1 × 15 min, 1 × 10 min, 2 × 5 min). Either a pool of sera from parasite-free animals or a pool of polyclonal sera derived from five sheep hyperimmune to H. contortus (1/500 in PBS-T/27 milk) was incubated overnight at 22–24 °C, washed, and incubated with anti-sheep total IgG coupled to horseradish peroxidase (1/75000 in PBS-T/27 milk) for 1 h at room temperature. The chemiluminescent development was performed with ECL Plus according to the manufacturer's instructions (Amersham Biosciences). Films exposed to the blots were densitometrically scanned using an ImageScanner, and matching was done by comparing the films of the blots with the DB71-stained membrane image and later with the master gel. Proteins were in-gel digested with trypsin (Roche Molecular Biochemicals) in 50 mm ammonium bicarbonate (Sigma). Before MALDI-TOF analysis, peptides were concentrated using ॖC18-ZipTips (Millipore) and eluted directly on the MALDI-target in 1 ॖl of a saturated solution of α-cyanohydroxycinnamic acid in 507 acetonitrile. Peptides were analyzed using a Voyager DE-STR MALDI-TOF mass spectrometer (Applied Biosystems) operated in reflectron mode at 20 kV accelerating voltage. Tandem MS measurements were performed on an electrospray ionization (ESI) quadrupole time-of-flight instrument (Q-Tof; Micromass Ltd., Manchester, UK) operating in positive ion mode and equipped with a Z-spray nano-ESI source. Nano-ESI needles were prepared from borosilicate glass capillaries (Kwik-FilTM, World Precision Instruments Inc., Sarasota, FL) on a P-97 puller (Sutter Instrument Co., Novato, CA). The needles were coated with a gold layer using an Edwards Scancoat sputter-coater 501 (at 40 mV, 1 kV, for 200 s). The capillary voltage was set at 1500 V; the cone voltage was 40 V. For the characterization of Hc15, the collision energy was optimized for individual peptides for optimal fragmentation. In all other cases, instead of nano-ESI needles a nano-LC system was coupled to the Q-TOF essentially as described in Ref. 15Meiring H.D. van der Heeft E. ten Hove J. de Jong A.P.J.M. J. Sep. Sci. 2002; 25: 557-568Crossref Scopus (222) Google Scholar. Peptide mixtures were delivered to the system using a Famos autosampler (LCPackings, Amsterdam, The Netherlands) at 3 ॖl/min and trapped on an AquaTM C18RP column (Phenomenex, Torrance, CA; column dimensions 1 cm × 100 ॖm inner diameter). After flow splitting down to 150–200 nl/min, peptides were transferred to the analytical column (PepMap; LC Packings, Amsterdam, The Netherlands; column dimensions 25 cm × 50 ॖm inner diameter) in a gradient of acetonitrile (17 per min). Fragmentation of eluting peptides was performed in data-dependent mode, and mass spectra were acquired in full-scan mode. For protein identification, Mascot software (www.matrixscience.com) was used for data base searches both for peptide mass fingerprinting and peptide sequence tagging. The scanned images were analyzed with Image Master two-dimensional v4.01 software (Amersham Biosciences). The observed molecular weight and isoelectric points (pI) of all the spots were calculated with protein markers (low molecular weight calibration kit, Amersham Biosciences) and pI markers (two-dimensional SDS-PAGE standards, Bio-Rad). Protein staining and immunological recognition of individual spots were quantified by calculation of spot volume after normalization of the image, using the total spot volume normalization method multiplied by the total area of all the spots (Image Master software). For the immunoblotting analysis, the image of the 2-min exposure was used for the quantification procedures. H. contortusEST sequences available from the dbEST division of GenBankTM (4843 on September 30, 2002) were clustered by SeqMan (Lasergene sequence analysis software, DNAstar) using a minimum match percentage of 90 and a minimum overlap of 20 bp. The alignments of the 342 clusters containing three or more ESTs were edited manually where necessary. The resulting 1876 clusters (of which 1253 contain a single EST) were annotated by running locally a Blastx (NCBI, cut-off p > 100) against the complete SwissProt and Trembl protein data bases. All EST clusters and all separate ESTs were subjected to a six-frame translation, and all peptide sequences longer than 60 amino acids were used for making a local data base that was searched by EMOWSE (16Pappin D.J.C. Hojrup P. Bleasby A.J. Curr. Biol. 1993; 3: 327-332Abstract Full Text PDF PubMed Scopus (1407) Google Scholar) (available from the EMBOSS package at www.hgmp.mrc.ac.uk/Software/EMBOSS). A tolerance of 0 was specified (effectively ± 0.5 dalton), and for cysteines modification by iodoacetamide was taken into account. Under optimized conditions of sample preparation, aiming at the prevention of proteolytic breakdown and limitation of artificial modifications, 224 Coomassie Blue-stained spots were detected in a 140-ॖg sample of H. contortus ESP (Fig.1). This complexity substantially exceeds previous estimations derived from one-dimensional SDS-PAGE and was even more apparent after silver staining, resulting in the automated detection of about 950 spots. Fig. 2shows a comparison of silver-stained gels of four batches of ESP obtained from different infections (see "Discussion"). Five Coomassie Blue-stained gels holding samples of the same batch of ESP were found by imaging software analysis to be nearly identical and were used for the reported experiments.Figure 2Silver-stained two-dimensional-SDS-PAGE gels of H. contortusESP. Four independent batches (70 ॖg) were run on a 12.57 two-dimensional gel (pI 3–10NL) as described under "Experimental Procedures." A, batch used for the mass spectrometry procedures (in Fig. 1). B,C, and D, three other independent batches. For comparison, some of the identified spots are marked in all four gels. Molecular mass markers are indicated in kDa.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 130 spots were judged to contain sufficient material for fingerprinting by MALDI-TOF mass spectrometry. Fingerprints were used for searching the GenBankTM non-redundant protein data base with Mascot software and allowed the identification of 61 spots of H. contortus origin. Because relatively few H. contortusentries are present in GenBankTM nr, an attempt was made to employ EST data for peptide mass fingerprint searching. H. contortus ESTs were clustered and searched by EMOWSE (as described under "Experimental Procedures") for matching the fingerprints. The top-score hits identified in 43 cases a contig matching one of the 61 spots identified by Mascot in GenBankTM nr. Additional EMOWSE hits, scoring within the range observed for the 43 confirmed Mascot hits, could represent significant identifications of proteins that are currently only present in the EST-derived data. To check for the reliability of these hits, selected spots were subjected to peptide fragmentation by LC-MS/MS to obtain sequences that could be used for searching both GenBankTM nr and EST data bases. In addition, manual comparison of peptide fingerprints of 12 spots, lacking LC-MS/MS data, revealed a very high similarity to the fingerprints of adjoining spots (marked as "identified by fingerprint similarity" in Supplementary Table S1 that includes all MW, PI, spot volume, and scoring data), which in most cases was confirmed by a matching EMOWSE top hit. These spots could represent post-translationally modified forms of the same protein. In this way, a total of 107 identifications were made from 102 spots, of which 62 were confirmed as top hit by EMOWSE searches of the EST-derived dataset (average score of 0.293, ranging from 0.132 to 0.556). In addition, for 14 spots confirmation by EMOWSE was obtained by lower ranking hits. The fingerprints of 8 spots with high EMOWSE scores (p > 0.250, average 0.345) lacked confirmation by other methods. Confidence in these hits was enhanced by a manual reinspection demonstrating that the fingerprints matched within 50 ppm mass accuracy to the exact calculated MWs of the predicted peptides of their respective EMOWSE hits (EMOWSE tolerates a less accurate MW range of ± 0.5 dalton). They are taken up in the supplementary table as preliminary identifications. In Fig. 3 a colored scheme is used for categorizing identified spots in functional groups. Immunoblotting results are also incorporated. Immunoblotting of two-dimensional gels in combination with sensitive chemiluminescence detection permits quantification of relative immunogenicity (included in Supplementary Table S1) of individual spots by taking the ratio between the density of a Coomassie Blue-stained spot and its chemiluminescence signal. Reproducible blots of ESP batch A were incubated with a pool of hyperimmune sera obtained from five sheep experimentally infected withH. contortus several times. Spots on the blot and the gel were matched by imaging software; the final image is shown in Fig.4. From 193 immune-recognized spots detected by image analysis, 52 have been identified above by mass spectrometry. Relative immunogenicity was observed to vary over a 2500-fold range (comparing spot 182 to spot 170) but is in fact larger because some high-density spots display no immunological detection at all, whereas several immunogenic spots cannot be detected by Coomassie Blue staining. This also reflects the specificity of immune recognition of the respective spots. TableI provides a list of protein identifications; full data can be found in Supplementary Table S1. Identifications that have exclusively been made by matches to ESTs have been annotated on the basis of BLAST analysis.Table IES proteins of H. contortus identifiedProtein descriptionNumber of IDs1-aID, identifications.Accession numberH. contortus ESP proteins Hc1521AAC47713,BF423205, BF059795, BF059790, BF066301, BF060216, BF060110 Hc245AAC47714, BF059884, BF422751 Hc241BF060098 Hc241BF059857 Hc242BF060283 Hc404AAC03562, BF060098 GA1- apical gut membrane protein21CAB60199, AAB01192,BE496641, BF422939Proteases Microsomal aminopeptidase✳ (H11–2, H11–4)4CAB57358, CAC39009 Metallopeptidase mep1✳6AAC31568, BE496726 Metallopeptidase mep2✳2AAC28740 Metallopeptidase mep1B✳2AAC03561, AI723449 Serine protease★3BF422843 Serine protease★2AI723535,BF422889, BF059836, BF423241, BF060014, BF060421 Serine protease★3BF422825 Aspartyl protease✳1CAA96571, AF079402Antioxidant enzymes Superoxide dismutase✳1Q27666, BG734232 Glutathione S-transferase★1BM138779Cytosolic enzymes Glutamate dehydrogenase4AAC19750,BG734171, BE496689, BG734187 Enolase★1BF422728,BF423327, BF060027, BF059800, BG734270 Lactate/malate dehydrogenase⋆1Ascaris suumBM281165, BM280602,BM283265, BG733747, BG733758, Amblyomma variegatumBM290097,C. elegans NP 504656 Aldolase★1BF642891,BM173804, BM138960, BM138894, BG123046 Triosephosphate isomerase⋆1Onchocerca volvulusAI381150,Ancylostoma caninumAW588250, Acipenser brevirostrumAF387818, Chrysops vittatusAAB48447 Aldehyde dehydrogenase★1BE228187Enzymes Peptidyl-prolyl-cis-transisomerase- type 3- cyclophilin★38858 Peptidyl-prolyl-cis-transisomerase- type 5- cyclophilin★1BF423227 Nucleoside diphosphate kinase★1BM139041, BM139053, BM138866Potential host proteins Complement C3 precursor1Mus musculusP01027, Sus scrofaAAG40565 Serpin1Bos taurusAAF23888 Serpin3Ovis ariesCAA33561, Bos taurusAAA50448Others Transthyretin-like domain★1BF423163, BF060057 Transthyretin-like domain, JC8.8 protein C. elegans protein★1BM138829,BM138926, BM138889, BM139325, BM139052, AW670763, BF059980, BM139192,BF060064, BM138977 F54D5.3 C. elegansprotein★1BF423000, BE228225, and 73 other H. contortus ESTs matching Y105C5B.5 C. elegansprotein★2AW670861, BG734233, BM173895 Y105C5B.5C. elegans protein★1BF642886, BF060428, BF423001,BF423135 OV39 antigen★2BF060234, BM138934, BM139330 Globin-like★1BF662828, BE496672, BE496783, BF060413 Total of IDs (total identified spots)107 (102)★, Only EST sequences available from H. contortus yet. ⋆, No sequence available from H. contortus. ✳, Not detected previously in H. contortusESP.1-a ID, identifications. Open table in a new tab ★, Only EST sequences available from H. contortus yet. ⋆, No sequence available from H. contortus. ✳, Not detected previously in H. contortusESP. Hc15, Hc24, and GA1 are the only proteins that have previously been shown to be ESP constituents (17Schallig H.D.F.H. van Leeuwen M.A.W. Verstrepen B.E. Cornelissen A.W.C.A. Mol. Biochem. Parasitol. 1997; 88: 203-213Crossref PubMed Scopus (72) Google Scholar, 18Jasmer D.P. Perryman L.P. McGuire T.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8642-8647Crossref PubMed Scopus (32) Google Scholar) by N-terminal peptide sequencing. These proteins will first be inspected in detail. Although for each a single gene has been cloned, we identified multiple spots for each of the three proteins (all of unknown function), hinting at sequence modifications (post-translational or in primary sequence). This was investigated in more detail for the spots identified as Hc15. Mascot and EMOWSE searches of MALDI-TOF fingerprints in combination with manual comparison of the fingerprints from this gel region identified 21 potential Hc15 protein spots distributed over a wide range of pI and MW (pI 5.53–7.04; 16.1–19.0 kDa) as indicated in Fig.3. Tandem mass spectrometry was pursued on these spots until high sequence coverage was obtained. All spots appear to be Hc15-related. In Fig. 5 the single published sequence of Hc15 (AAC47713) is taken as a basis, and all sequenced parts of the individual spots are shaded. Amino acid substitutions, all of which can be explained by single nucleotide changes, are observed in 10 of 21 spots. One to 5 residues are substituted out of a set restricted to 6 positions (Fig. 5, shaded in black) and occur in 9 different combinations. Four of 6 (T46A, R64P, T73A, and V90I) are also observed to be encoded by one or more of the 19 ESTs matching Hc15 and are thus confirmed to be present at the protein level in ESP. Two additional substitutions (H47R and N72S), detected by de novo sequencing using the MS data, were not found in ESTs. Spectra of six spots contained a 1362-Da mass peak, the full sequence (SGNQVMFENINK) of which does not correspond to a tryptic fragment and therefore possibly represents the N terminus (Fig. 5). This N terminus exactly matches the end of an 11-amino acid deletion (between Glu-20 and Gly-30) in three ESTs (BF059795, BF422966, BF423305). Both in these three ESTs and in all other sequences, signal peptide cleavage is predicted to occur after Gly-19 (SignalP). The predicted N-terminal tryptic peptide of AAC47713 (ESQLNTK) and of the other 16 ESTs was not observed in any of the spots, suggesting alternative cleavage at the N terminus at Gly-30. A total of 17 of the 19 available Hc15 ESTs contain the stop codon and, as concluded from an alignment of these EST sequences, the 35-amino acid C-terminal tryptic peptide seems to be the most conserved part of the protein. Thus, spots 188 and 190 on the one hand and spots 161, 162, 165, and 167 on the other hand are almost fully sequenced by MS/MS and predicted to have an identical number of amino acids but, nevertheless, display a ∼2000-Da mass difference. Possibly post-translational modifications are involved in such differences. Collectively, the residue substitutions and alternative N-terminal sequences result in considerable variation among Hc15 proteins. A direct relationship between sequence variation and immune recognition, as measured by the relative immunogenicity described in Fig.6, is far from obvious. Only 2 of the 10 spots in which substitutions have been found are recognized by hyperimmune serum (Fig. 5, spots 177 and 189), and even spots in which no sequence variation is detected by MS/MS display a 5-fold difference in immunogenicity (e.g. spots 180 and 183) or are not detected at all (e.g. prominent spots 175 and 193). Variation in relative immunogenicity is even more pronounced within the groups identified as Hc24-like (9 spots) and GA1-like (21 spots). For the Hc24 group a 32-fold difference was observed (comparing spot 128 to 131), whereas one of nine was not recognized at all (spot 119). Similar to the Hc15 group, spots are distributed over a wide pI and MW range (4.80–6.71; 26.2–34.3 kDa). Spots of the GA1 group are located in a more restricted area (see below) accommodating many other, sometimes co-migrating, spots (more clearly demonstrated after silver staining). This increases fingerprint complexity and might explain the low, but significant, Mascot scores of a number of GA1 identifications (Table S1). In addition, low scores may point at considerable sequence modification between spots and the published GA1 sequence, a feature that can only be clarified by an extensive MS/MS approach as used above for Hc15. GA1 protein is a 92-kDa membrane-associated polyprotein from which p46GA1 and p52GA1 subunits are released into solution after cleavage by glycosylinositol-specific phospholipase C (18J
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